Wei, Xiaoping , Gou, Xiaoping , Yuan, Tong , Russell, Scott D. .
Highly efficient plant regeneration and Agrobacterium-mediated transformation of Plumbago.
Plumbago zeylanica is a unique model for studying flowering plant gametogenesis, heterospermy, and preferential fertilization, yet understanding the control of related molecular mechanisms is impossible without an efficient, reproducible and stable genetic transformation system. We found three key factors to successful tissue culture in this plant: (1) the tissue source of explants, (2) growth regulator types and combinations, and (3) culture conditions. Hypocotyl, stem and petiole were most responsive as explants for shoot regeneration. Media containing coconut milk, with or without the addition of adenine, greatly increased shoot regeneration frequency. By reducing light intensity, the browning of callus cultures was minimized and shoot regeneration was therefore enhanced. The highest shoot regeneration frequency was achieved using hypocotyl segments cultured on an MS basal medium supplemented with BA 2.0 mg/l, NAA 0.75 mg/l, adenine 50 mg/l and 10% (v/v) coconut milk (CM) under subdued light at 25 ± 2°C; under these conditions, each hypocotyl segment produced over 30 shoots, which arose primarily through direct organogenesis along the cut edge of the hypocotyl segment after 3 weeks of culture. Using this tissue culture protocol, reporter gene GUS under the constitutive CaMV 35S promoter was introduced into P. zeylanica cells of petiole segments and axillary buds with A. tumefaciens strains AGL1, EHA105, GV3101 and LBA4404. Transient expression was observed in all recipient tissue types for a period of 2 weeks. Stable transgenic calli originating from petiole were obtained in approximately 10% of petioles. Chimeric transgenic shoots could be easily obtained from transformed axillary buds, but the most stable results were from petiole. Thus, our protocol met the critical factors affecting gene transformation efficiency in P. zeylanica and are suitable for production of stable transgenic plants.
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1 - University of Oklahoma, Department of Botany and Microbiology, 770 Van Vleet Oval, Norman, Oklahoma, 73019, USA
Presentation Type: Array
Location: Salon C, D & E - Gov Ballroom/Hilton
Date: Tuesday, August 16th, 2005
Time: 12:30 PM